DNA topoisomerases are enzymes that change the topological state of DNA. They are involved in several crucial cellular processes such as replication, transcription, chromosome segregation, and chromosome condensation. More important, they have become the focus of study as a target of novel anti-bacterial and chemotherapeutic agents. The understanding of the structure of topoisomerases promises not only to further our understanding of proteins that interact with DNA and modify its topological properties, but also to provide important information to aid in the design of new therapeutic agents. The long term goal of this proposal is to understand the mechanism of action of type I topoisomerases in atomic detail. Two type I topoisomerases have been identified in Escherichia coli, DNA topoisomerase I and III. The investigators have solved the structure of the two enzymes and their structures suggest an enzyme-bridged mechanism that may apply to all type 1-5' topoisomerases and that has interesting parallelism with the mechanism of type II topoisomerases. The investigators have proposed a detailed mechanism for the cleavage/religation reaction that they are currently testing by mutagenesis. Other studies have allowed identification of two putative DNA binding sites by protein crystallography. The specific aims for this proposal are: i) to refine the structure of the intact and active E. coli DNA topoisomerase III to high resolution (2.4 A or better), ii) to study the interactions of E. coli DNA topoisomerases I and III with DNA, iii) to identify and characterize the structural and chemical determinants of catalytic activity and to elucidate their specific role in the reaction, and iv) to characterize and solve the structure of different domains of E. coli DNA topoisomerase I. The work is based on a combination of molecular biology and biochemical methods to produce and characterize the proteins that we require for our work, and x-ray crystallography to solve their structures to high resolution.